CAPS Bioscience Strand

7501 West 149th Terrace

Overland Park, Kansas 66223

ekessler@bluevalleyk12.org

.

Mentors: Dr. Aron Fenton & Qingling Tang

References

Defossiling Fuel Article

Synechocystis & PK Article

Synechococcus & PK Article

Metabolic Network of Synechocystis

caps Website

Hawaii’s plasmid

Rabbit Muscle Pyruvate Kinase

Yeast Pyruvate Kinase

Registry primer design

Registry 3A

Safety Manual

Middle School Slideshow

Microryza Project Page

THE CLEAN GREEN CYANOMACHINE Microbial Production of a Sustainable Energy Source (Alkanes) through Overexpression of Pyruvate Kinase Alec Adams, Corinne Andresen, Muriel Eaton, Dylan Geick, Austin Jacobson, Amy McClain, & Mason Wilkinson

Humans are dependent on carbon for food and energy. Industrialization led to an increase in population that was powered by the discovery and combustion of fossil fuels. Resultant carbon dioxide emissions threaten to destabilize our climate, while dependence on oil subjects its users to the economic and political instability associated with volatile oil supplies and prices.

Chasses & Metabolic Pathway Cyanobacteria are important to global CO2 fixation, O2 recycling, and N2 fixation. They are the ancestors of the chloroplast, algae, and terrestrial plants, and are thought to be responsible for the original oxygenation of Earth’s atmosphere and for the vast oil deposits found across globe. The cyanobacterial species Synechocystis PCC 6803 and Synechococcus PCC 6801 have been chosen for our current work for the following reasons:

• Safe and easy to use, and amenable to genetic manipulation • Diverse metabolic network and genome fully sequenced • They can live on waste water and are not in competition with

food production

Pyruvate kinase catalyzes the transfer of a phosphate group from PEP to ADP, to produce one molecule of pyruvate and one ATP. This is the final step of the glycolytic pathway. This reaction is potentially a key regulatory step in engineering of the carbon metabolism in cyanobacteria. Specifically, we hypothesize that increased pyruvate production, through overexpression of pyruvate kinase should enhance the production of lipids and alkanes.

Problems & Solutions Next school year, we plan to finish making and characterizing our two parts. We will also expand upon our current Tinker Cell model in an effort to make predictions regarding pyruvate kinase and other key regulators in the pathways responsible for alkane production. If time permits, we may test combinations of cyanobacterial promoters (see adjacent reference) and ribosomal binding sites with our parts. Finally, we hope to expand our community outreach with the addition of a BioBuilder workshop for teachers taught by our instructors and next year’s iGEM participants.

Future

During the first week of June, we hosted a four-day bioscience middle school camp where they learned to:

• Use micropipettes, centrifuge, and vortex machine • Extract DNA from their own check cells • Perform sterile technique and streak plating • Transform bacteria using the heat-shock method • Perform gel electrophoresis of DNA

Outreach

Educational Innovation This year, the team turned to crowdfunding to supplement the funds associated with this projects, and for minimizing the costs associated with travel for those students interested in participating in this iGEM Jamboree. We used the Microryza crowdfunding website which specializes in raising money for science research projects. Our project was the first education oriented science project on their site. The University of Washington and Purdue University iGEM teams have recently posted their 2013 iGEM projects on this site. Here are some general statistics from our funding:

• We raised a total of $2355 from 28 backers • Donations ranged from $5 to $1000 • Most gifts were $25 with the average being $84 • All but four of the donations were $75 or less

1. S D. Savage, J. Way, and P. Silver (2008) “Defossiling Fuel: How Synthetic Biology Can Transform Biofuel Production” ACS Chemical Biology. 3(1), 13-16.

2. H. Knoop, Y. Zilliges, W. Lockau, and R. Steuer (2010) “The Metabolic Network of Synechocystis sp. PCC 6803: Systemic Properties of Autotrophic Growth” Plant Physiology. 154, 410–422.

3. V. Knowles, C.Smith, C. Smith, and W. Plaxton (2001) “Structural and Regulatory Properties of Pyruvate Kinase from the Cyanobacterium Synechococcus PCC 6301” J. Biol. Chem. 276, 20966-20972.

4. Vicki L. Knowles and William C. Plaxton (2003) “From Genome to Enzyme: Analysis of Key Glycolytic and Oxidative Pentose Phosphate Pathway Enzymes in the Cyanobacterium Synechocystis sp. PCC 6803” Plant Cell Physiol. 44(7), 758–763.

Wiki References

During our work on this project, our team took the following basic safety precautions:

• Lab coats, goggles , and gloves were worn, when necessary, for personal safety and for minimizing contamination

• Students were always supervised when in the laboratory • Contaminated materials were autoclaved before disposal • Food and drink were not consumed within the laboratory

Safety

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Alkanes are a significant component of gasoline, diesel, and jet fuel. Metabolic engineering of microbes promise a means of reducing our dependence on non-renewable fuels while replacing it with fuel derived from a carbon-neutral source.

PROJECT OVERVIEW

Synechocystis PCC 6803

Synechocystis PCC 6803

Synechococcus PCC 6801

Synechococcus PCC 6801

Characterizations

1) Assay Pyruvate Kinase Activity

2) Lipid Profile using Gas Chromatography

3) Alkane Profile using Gas Chromatography

BBa_K977000 or BBa_K977001

Rabbit Muscle or Yeast PK Gene RBS Stop Stop

Pyruvate PEP CO2 Lipids ALKANES

Methods Standard protocols were used for plasmid mini-preps, assembly of parts, and transformation of cells. In order to construct our parts, it was necessary to perform PCR Site-Directed Mutagenesis to remove two restriction sites from within each of our parts. We also had to PCR the standard BioBrick prefix and suffix on the flanks of each part.

Pyruvate Kinase

Metabolic Pathway

Pyruvate Kinase Device

Plasmid Constructs with Parts Chasses

3A Assembly

PCR BioBrick Stadards

caps iGEM Wiki

Instructors: Eric Kessler, Joe Whalen, & Kelley Tuel

Cyanobacterial Promoters Article